Podocytes are highly differentiated epithelial cells in the kidney glomerulus that possess extensively branched cell processes, interdigitating into the unique foot processes and slit diaphragms which function as key components of the filtration barrier. Genetic mutations in ACTN4 disrupt cytoskeletal structure of the podocytes and have been linked to several glomerular diseases. However, the normal function of ACTN4 in podocytes as well as the mechanism underlying disease-causing ACTN4 mutations is not clearly understood. Based on our preliminary data, we hypothesize that ACTN4 plays a role in both the nucleus and the cytoplasm and that ACTN4 is capable of modulating transcriptional activity of nuclear receptors in the nucleus of podocytes. The Specific Aims are: 1) To characterize the role of ACTN4 in conditionally immortalized human podocytes (HPCs). We will knockdown endogenous ACTN4 and determine the effect on the expression of podocyte marker genes. We will also establish cell lines expressing ACTN4 to establish direct binding to selected target genes and globally identify its associated genes by ChIP (chromatin immunoprecipitation)-on-ChIP analyses. 2) To dissect the mechanisms by which ACTN4 and nuclear hormone receptors regulate the expression of nephrin. We will determine the sequence determinants within the nephrin promoter that are responsive to ACTN4, nuclear receptors, and their ligands by transient transfection reporter assays. We will verify these data by ChIP assays and delineate histone modification patterns in response to hormones. We will further test whether known human disease-linked PPAR mutations have an effect on podocytes. 3) To explore the role of FSGS- causing ACTN4 mutations on normal podocyte behavior and gene expression. Once we have established the normal functions of ACTN4 in Aims 1 & 2, we will examine the disease-linked ACTN4 mutants and their role in transcriptional regulation, histone modifications, and hormone response using the nephrin promoter as a model. We anticipate that our studies on ACTN4 and its interacting proteins will uncover a previously underappreciated nuclear role for ACTN4 that is critical for podocyte functions and may have future therapeutic implications in podocyte diseases. PUBLIC HEALTH RELEVANCE: One of the key functions of the kidney is to remove toxins and metabolic waste while preventing proteins larger than albumin from entering the urine. This process is mediated by highly specialized cells known as podocytes that produce critical components of the filtration barrier in glomeruli. Genetic mutations in a known cytoskeletal structural protein, ACTN4, have been linked to several glomerular diseases. We have identified a novel function of ACTN4 as a transcriptional coactivator in the nucleus that modulates the transcription of several hormone-sensitive genes. We will investigate the details of this nuclear function. We anticipate that our studies will uncover a previously underappreciated role for ACTN4 that is critical for podocyte functions and may have future therapeutic implications in kidney diseases.